The long-term objective of this study Is to understand the factors regulating osteoblastic cell calcium. It is becoming increasingly evident that changes In this ion are involved in the mechanism of action of several resorptive agents and that an understanding of the various calcium regulatory systems will aid in the development of potential ways to control pathological changes in bone metabolism occurring systemically or locally. Specifically, it is proposed in osteoblastic cells isolated from the calvaria of newborn rats and In the osteoblastic human osteosarcomal cell lines, SaOS-2 and G292, to: 1. to further study the nature of the regulation of mobilization of calcium from intracellular pools with particular emphasis on the role of guanine trisphosphate and Its possible interaction with inositol phosphates such as 1(1,4,5)p3; 2. to delineate the nature of regulation of phosphoinositide metabolism and cytosolic calcium levels by the bone resorptive agent and mitogen, epidermal growth factor (EGF), in order to better compare and contrast its effects with other bone resorptive agents such as thrombin; 3. to delineate the nature of regulation of phosphoinositide metabolism by calcium via experimentation with calcium channel antagonists, an ionophore and extracellular calcium levels; 4. to study the electrophysiological properties of these cells with emphasis on membrane potential and potassium, sodium and calcium currents; 5. to study the effects of EGF and thrombin on these electrophysiological properties; 6. to study the effects of inositol phosphates on the electrophysiological properties defined in aims 4 & 5; 7. to study the role of tyrosine kinase activity in the regulation of osteoblastic cell calcium, phosphoinositide metabolism as well as proliferation In response to EGF. Cytosolic calcium regulation will be studied with the fluorescent dye, Fura-2, and phosphoinositide metabolism in tritiated -inositol labelled cells by means of high performance liquid chromatography. Cell proliferation will be assessed by incorporation of tritiated thymidine. The electrophysiological properties will be studied with patch-clamp techniques. In general, the studies proposed here should provide more In depth information on the intracellular and plasma membrane regulatory mechanisms involved in the control of osteoblastic cell calcium with emphasis. on the possible diversities of these pathways involved upon activation by agents as potentially different as EGF and thrombin.